Malaria is a devastating disease with enormous negative impact on global health. An efficacious vaccine for malaria would be a huge boon for the >40% of humanity with regular exposure to Plasmodium infected mosquitoes. Although a partially protective vaccine (RTS, S) is in phase III trials, subunit vaccines to induce sterilizing liver-stage specific immunity in humans have proven difficult to develop. In contrast, it has been known for over 30 years that radiation attenuated sporozoite (RAS) immunization delivered by mosquito bite can provide sterile immunity to humans. Studies in mice show clear relevance of T cells, particularly CD8 T cells, for sterilizing immunity to liver stage Plasmodium infection. Importantly, human clinical trials of immunization with RAS are underway. In addition, much recent effort has been directed at developing genetically attenuated parasite (GAP) sporozoite vaccines that may have better safety profiles and provide more reliability than RAS. GAP vaccines are also entering initial human clinical trials. Finally, recent data demonstrate that sporozoite infections under chloroquine drug cover can also generate substantial protective immunity in mice and humans. However, it is unknown which of these whole-sporozoite immunization regimens is most effective at generating protective T cell responses. Given the hurdles inherent in deploying a sporozoite based vaccine in the developing world, pre-clinical insight into the best T cell stimulating vaccines could provide a rational basis to focus evaluation of candidate platforms and substantially accelerate progress toward an efficacious malaria vaccine. As detailed in our recent publication in PLOS Pathogens and highlighted in the preliminary data, we recently applied a surrogate activation marker approach, developed in our laboratory, to track the total T cell response to RAS vaccination in inbred and outbred mouse strains. We now propose to use this approach to characterize the T cell response as well as antibody responses against the spectrum of candidate whole parasite vaccines in order to identify the most potent vaccines in preclinical studies. We will address this knowledge gap through the following specific aims: Specific Aim 1. Compare the total CD8 T cell response and protective immunity in RAS versus GAP immunized inbred and outbred hosts. Specific Aim 2: Apply a surrogate activation marker approach to dissect the role of CD4 T cells in antimalarial protection after whole-sporozoite immunization. Specific Aim 3: Characterize T cell and antibody responses and protective immunity to live sporozoite infection under chloroquine drug cover in inbred and outbred hosts. Specific Aim 4: Evaluate optimal whole-parasite vaccines for the ability to elicit protective T cell and antibody responses via translationally relevant routes of immunization PUBLIC HEALTH RELEVANCE: Plasmodium infections cause 300-500 million cases of malaria each year and have a devastating impact on global human health, with particularly high mortality in children living in sub-Saharan Africa. Currently, whole parasite sporozoite vaccines can be generated by irradiation, deletion of critical genes or by antimalarial drugs administered during infection. The goal of the current proposal is to directly compare the ability of each of these vaccine platforms to elicit protective T cells or antibodies and immunity to liver or blood stage infection in order to identify the most effective whole parasite vaccine.